Recent advances in optical imaging and stimulation technology have allowed the merging of neurophysiological and neuroanatomical findings. In particular, in mammalian cerebral cortex, physiological studies have focused mostly on single neuron properties and the large-scale spatial distribution of these properties in cortex (topography); whereas, anatomical studies have focused mostly on neuronal structure including intrinsic and extrinsic areal projections-both between and across laminae, and to and from the thalamus. The main organizational feature of the auditory cortex (ACX) is the tonotopic axis that consists of a best frequency (BF) gradient over the rostro-caudal axis in most mammalian species. This organization has predominantly been probed with large-scale intrinsic imaging or single/multi-electrode electrophysiological recordings. Recent in vivo single cell imaging experiments revealed a large heterogeneity of this organization on small spatial scales. While these imaging studies showed that there is heterogeneity in frequency tuning, cells did show similarities in the subthreshold response properties. Other recent in vitro studies utilizing laser scanning photostimuluation techniques have observed local organization of functional connectivity patterns in ACX. These studies have found tonotopically-aligned asymmetries and unique columnar arrangements of connectivity in vitro using coronal and thalamocortical slices. By bringing together both single cell imaging and photostimulation, our aim here is to work towards merging neurophysiological and functional neuroanatomical findings by focusing on small-scale intralaminar functional connectivity both in vivo and in vitro. Preliminary in vivo imaging experiments for this proposal are consistent with the existence of local microcircuits. Such microcircuits could underlie local computational function and might be replicated as modules in A1. Where do these circuits originate? Are they an emergent property of layer 2/3 or do they form in layer 4? To address these questions we propose to characterize these local microcircuits in layer 2/3 (Aim 1) and the laminar origin of these circuits (Aim 2). We address these questions by using a combination of in vivo and in vitro 2-photon imaging coupled with photostimulation. Specifically, we propose to address intralaminar micro-organization of layer 2/3 and layer 4 in ACX. We hypothesize that many functionally relevant circuits are present in layer 2/3 de novo and that the fan out of ascending connections from layer 4 is responsible for the greater tonotopic heterogeneity that has been observed using imaging versus traditional electrophysiological techniques. PUBLIC HEALTH RELEVANCE: Understanding normal adult small-scale functional organization will allow for a template that can potentially be used to evaluate therapies for disorders that affect auditory cortex, particularly autism, tinnitus and schizophrenia. Description of small-scale functional organization is a first step to understanding its physiological role.